This invention relates generally to gene therapy. More particularly, the present invention relates to a synthetic polynucleotide and to a method for selectively expressing a protein in a target cell or tissue in which at least one existing codon of a parent polynucleotide encoding the protein has been replaced with a synonymous codon. The invention also relates to production of virus particles using one or more synthetic polynucleotides and the method according to the invention.
While gene therapy is of great clinical interest for treatment of gene defects, this therapy has not entered into mainstream clinical practice, at least in part because selective delivery of genes to target tissues has proven extremely difficult. Currently, viral vectors are used, particularly retroviruses and adenovirus, which are to some extent selective. However, many vector systems are by their nature unable to produce stable integrants and some also invoke immune responses thereby preventing effective treatment. Alternatively, xe2x80x9cnakedxe2x80x9d DNA is packaged in liposomes or other similar delivery systems. A major problem to be overcome is that such gene delivery systems themselves are not tissue selective, whereas selective targeting of genes to particular tissues would be desirable for many disorders (e.g., cancer therapy). While use of tissue specific promoters to target gene therapy has been effective in some animal models it has proven less so in man, and selective tissue specific promoters are not available for a wide range of tissues.
The current invention has arisen unexpectedly from recent investigations exploring why papillomavirus (PV) late gene expression is restricted to differentiated keratinocytes. In this regard, it is known that PV late genes L1 and L2 are only expressed in non-dividing differentiated keratinocytes (KCs). Many investigators including the present inventors have been unable to detect significant PV L1 and L2 protein expression when these genes are transduced or transfected into undifferentiated cultured cells, using a range of conventional constitutive viral promoters including retroviral long terminal repeats (LTRs) and the strong constitutive promoters of CMV and SV40.
PV L1 mRNA can however be efficiently translated in vitro using rabbit reticulocyte cell lysate, suggesting that there are no cellular inhibitors in the lysate interfering with translation of L1. The major difference between the in vitro and in vivo translation systems is that L1 comprises the dominant L1 mRNA in in vitro translation reactions, while it constitutes a minor fraction among the cellular mRNAs in intact cells.
In vivo, PV late proteins are not produced in undifferentiated KC. However, they are expressed in large quantity in highly differentiated KC. The mechanism of this tight control of late gene expression has been poorly understood, and searches by many groups for KC specific PV gene transcriptional control proteins have been unrewarding.
Blockage of translation of L1 mRNA in vivo has been attributed to sequences within the L1 ORF (Tan et al. 1995, J. Virol. 69 5607-5620; Tan and Schwartz, 1995, J. Virol. 69 2932-2945). By using a Rev and Rev-responsive element of HIV, such inhibition could be overcome (Tan et al. 1995, supra) . Accordingly, the inventors examined whether removal of putative xe2x80x9cinhibitory sequencesxe2x80x9d in the L1 ORF would allow production of L1 protein in undifferentiated cells. Deletion mutagenesis of BPV L1 to remove putative inhibitory sequences and expression of resultant deletion mutants in CV-1 cells revealed surprisingly that despite expression of L1 mRNA, L1 protein could not be detected.
In view of the foregoing, it has been difficult hitherto to understand how papillomaviruses produce large amounts of L1 protein in the late stage of their life cycle using this apparently xe2x80x9cuntranslatablexe2x80x9d gene. The present inventors have discovered the mechanism by which L1 protein is expressed in the late stage of the life cycle of this virus, and have also discovered a method of general application whereby polynucleotides can be designed or modified in order to effect selective expression of a protein in a target cell or tissue.
Accordingly, in one aspect of the invention, there is provided a method of constructing a synthetic polynucleotide from which a protein is selectively expressible in a target cell of a mammal, relative to another cell of the mammal, said method comprising:
selecting a first codon of a parent polynucleotide for replacement with a synonymous codon which has a higher translational efficiency in said target cell than in said other cell; and
replacing said first codon with said synonymous codon to form said synthetic polynucleotide.
Preferably, said first codon and said synonymous codon are selected by:
comparing translational efficiencies of individual codons in said target cell relative to said other cell; and
selecting said first codon and said synonymous codon based on said comparison.
A translational efficiency of a codon may be determined by any suitable technique. In a preferred embodiment, the translational efficiency of a codon is measured by:
introducing into said target cell and into said other cell, a synthetic construct comprising a reporter polynucleotide fused in frame with a tandem repeat (e.g. 2, 3, 4, 5, 6, or 7 or more) of said individual codon, wherein said reporter polynucleotide encodes a reporter protein, and wherein said synthetic construct is operably linked to a regulatory polynucleotide; and
comparing expression of said reporter protein in said target cell and in said other cell to determine the translational efficiency of said individual codon in said target cell relative to said other cell.
Preferably, the above method is further characterized by:
introducing the synthetic construct into a progenitor cell of a cell selected from the group consisting of said target cell and said other cell; and
producing said target cell from said progenitor cell, wherein said cell contains said synthetic construct.
Suitably, this method is further characterized by:
introducing the synthetic construct into a progenitor cell of a cell selected from the group consisting of said target cell and said other cell; and
growing an organism or part thereof from said progenitor cell, wherein said organism comprises said cell containing said synthetic construct.
The above method may be further characterized by the step of introducing the synthetic construct into an organism or part thereof such that said synthetic construct is introduced into said target cell or said other cell.
Preferably, said synonymous codon corresponds to a reporter construct from which the reporter protein is expressed in said target cell at a level that is at least 110%, preferably at least 200%, more preferably at least 500%, and most preferably at least 1000%, of that expressed from the same reporter construct in said other cell.
In an alternate embodiment, the translational efficiency of a codon is compared by measuring the abundance of an iso-tRNA corresponding to said individual codon in said target cell relative to said other cell.
Preferably, said synonymous codon corresponds to an iso-tRNA which is in higher abundance in the target cell relative to said other cell.
Preferably, selecting said first codon and said synonymous codon comprises:
measuring abundance of different iso-tRNAs in said target cell relative to said other cell; and
selecting said first codon and said synonymous codon based on said measurement, wherein said synonymous codon corresponds to an iso-tRNA which is in higher abundance in said target cell than in said other cell.
Advantageously, said synonymous codon corresponds to an iso-tRNA that is present in said target cell at a level which is at least 110%, preferably at least 200%, more preferably at least 500%, and most preferably at least 1000%, of the level that is present in said other cell.
Alternatively, the step of selecting may be characterized in that a synonymous codon according to the invention is selected from the group consisting of (1) a codon used at relatively high frequency by genes, preferably highly expressed genes, of a said target cell or tissue, (2) a codon used at relatively high frequency by genes, preferably highly expressed genes, of the mammal, (3) a codon used at relatively low frequency by genes of a said one or more other cells or tissues, and (4) a codon used at relatively low frequency by genes of another organism.
The step of selecting may be characterized in that a first codon according to the invention is selected from the group consisting of (a) a codon used at relatively high frequency by genes, preferably highly expressed genes, of a said one or more other cells or tissues, (b) a codon used at relatively low frequency by genes of a said target cell or tissue, (c) a codon used at relatively low frequency by genes of the mammal, and (d) a codon used at relatively high frequency by genes of another organism.
In a preferred embodiment, the method further includes the step of selecting the first codon and the synonymous codon such that said protein is expressed from said synthetic polynucleotide in said target cell or tissue at a level which is at least 110%, preferably at least 200%, more preferably at least 500%, and most preferably at least 1000%, of that expressed from said parent polynucleotide in said target cell or tissue.
Preferably, the other cell is a precursor cell of the target cell. Alternatively, the other cell may be a cell derived from the target cell.
In another aspect, the invention provides a synthetic polynucleotide constructed according to any one of the above methods.
Suitably, said synonymous codon(s) are selected from the group consisting of gca (Ala), cuu (Leu) and cua (Leu), when said target cell is a differentiated cell, and more preferably when said target cell is a differentiated keratinocyte.
Synonymous codons for higher level expression of a protein in an undifferentiated cell, preferably an undifferentiated keratinocyte, are suitably selected from the group consisting of cga (Arg), cci (Pro) and aag (Asn).
Synonymous codon(s) are preferably selected from the group consisting of aga (Arg), cgg (Arg), tgc (Cys), gga (Gly), ggc (Gly), ccg (Pro), cga (Pro), aca (Thr), acg (Thr), and act (Thr), when said target cell is an undifferentiated cell, and preferably an undifferentiated epithelial cell.
Suitably, the first codon is selected from the group consisting of agg (Arg), tgt (Cys), ggg (Gly), ggt (Gly), ccc (Pro), cct (Pro), and acc (Thr), when said target cell is an undifferentiated cell, and preferably an undifferentiated epithelial cell.
In yet another aspect, the invention resides in a method for selectively expressing a protein in a target cell or tissue of a mammal, said method comprising:
replacing a first codon of a parent polynucleotide encoding said protein with a synonymous codon to produce a synthetic polynucleotide having altered translational kinetics compared to said parent polynucleotide, such that said protein is expressible in said target cell, but such that said protein is not substantially expressible in another cell of the mammal; and
introducing into a cell selected from the group consisting of said target cell and a precursor of said target cell, said synthetic polynucleotide operably linked to a regulatory polynucleotide. The protein is thereby selectively expressed in said target cell.
Preferably, said synonymous codon has a higher translational efficiency in said target cell than in said other cell.
In yet another aspect, the invention provides a method of expressing a protein in a target cell from a first polynucleotide, said method comprising:
introducing into a cell selected from the group consisting of said target cell and a precursor of said target cell, a second polynucleotide encoding an iso-tRNA, wherein said second polynucleotide is operably linked to a regulatory polynucleotide, and wherein said iso-tRNA is normally in relatively low abundance in said target cell and corresponds to a codon of said first polynucleotide. The protein is thereby expressed in the target cell.
In a further aspect, the invention extends to a method of producing a virus particle in a cycling eukaryotic cell, wherein said virus particle comprises a protein necessary for assembly of said virus particle, and wherein said protein is not expressed in said cell from a parent polynucleotide at a level sufficient to permit virus assembly therein, said method comprising:
replacing a first codon of said parent polynucleotide with a synonymous codon to produce a synthetic polynucleotide having altered translational kinetics compared to said parent polynucleotide, such that said protein is expressible from said synthetic polynucleotide in said cycling eukaryotic cell at a level sufficient to permit virus assembly therein; and
introducing into a recipient cell selected from the group consisting of said cycling eukaryotic cell and a precursor of said cycling eukaryotic cell said synthetic polynucleotide operably linked to a regulatory polynucleotide, wherein said recipient cell comprises said protein necessary for assembly of said virus particle. The protein is thereby expressed and said virus particle is produced in said cycling eukaryotic cell.
In yet a further aspect of the invention, there is provided a method of producing a virus particle in a cycling cell, wherein said virus particle comprises at least one protein necessary for assembly of said virus particle, wherein said protein is not expressed in said cell from a parent polynucleotide at a level sufficient to permit virus assembly therein, and wherein a codon of said parent polynucleotide is rate limiting for the production of said protein, said method comprising introducing into said cell a polynucleotide capable of expressing therein an iso-tRNA specific for said codon. The virus particle is thereby produced in the cycling cell.
The invention also includes cells, vectors, and viruses made by any of the methods described above, and pharmaceutical compositions comprising one or more such cells, vectors, and viruses together with a pharmaceutically acceptable carrier.